During the first two years of this project we have developed methodology for the growth of human colonic cacinoma on feeder layers of confluent fibroblasts. This methodoloy has resulted in the long-term culture of 31/37 attempted sppcimens. We have identified 3 morphological patterns of growth in primary specimens which appear to be associated wth subpopulations of cells which have been isolated from 4 different carcinomas. These subpopulations have a wide range of malignant potential as assessed by tumorigenicity in nude mice and other in vitro criteria normally associated with malignancy. In this renewal application we propose to improve our culture methods by determining the optimal cell type for feeder layers, identifying growth factor(s) produced by feeder layers and increasing inocula of cultures by employing purified rather than unseparated malignant cells containing other cell types. These improvemnts may eventually lead to the development of an in vitro screening system of possible therapeutic modes for individual patients and will improve the chances of isolating subpopulations of cells from individual tumors. Isolated subpopulations of cells will be extensively characterized in order to determine whether they developed by reversible differentiation processes or by irreversible mutation. Determination of the route by which subpopulations of cells with different malignant potential are produced in individual tumors will have important effects on the future development of therapeutic strategies for colon cancer.